The emergence of multidrug resistance (MDR) in Acinetobacter baumannii has resulted in the designation of this Gram negative bacterium as a priority in Infectious Diseases. The long-term goal of this work is to understand the molecular basis for antibiotic resistance in A. baumannii and the role of mobile genetic elements in the evolution of resistance. Genes encoding antibiotic resistance determinants are frequently associated with mobile genetic elements in A. baumannii including insertions sequences (IS) and in MDR strains many resistance genes are co-located in a laterally transferred 'resistance island'(RI). Genome sequencing of closely related strains from the same hospital revealed variation in the complement of resistance genes that corresponded with differences in antimicrobial susceptibility pattern. The role of mobile genetic elements and other genetic changes in the evolution of antibiotic resistance will be explored through characterization of collections of strains from three recent A. baumannii outbreaks in unprecedented detail. The strategy is designed to identify genotypic variation among extensively drug resistant strains and phenotypic variation among genotypically similar or identical strains. These strain collections will enable consideration of genetic changes over time periods of weeks to months that are associated with changes in antibiotic resistance. Molecular sequencing typing will be used to establish broad patterns of relatedness among strains and to identify instances of variation in resistance phenotype over time by clone type. Whole-genome sequencing and gene expression analysis will provide detailed information about the presence, organization, and regulation of genes associated with resistance. The rate of mobilization of IS elements and changes in the RI will be determined through phylogenetic analysis. Although specific genes are known to contribute to resistance to certain antibiotics, it is also likely that high level resistance is the result of a combination of factors including drug inactivation, efflux, and target site modification. Analysis of diverse stains will lead to identification of alternative gene sets that lead to resistance. The outcome of this study will be a better understanding of how multidrug resistance evolves in a clinical setting. It will also result in a more complete view of the mechanisms that lead to antibiotic resistance in A. baumannii, which will assist in development of molecular diagnostic assays to inform selection of the most appropriate therapeutic regimen. PUBLIC HEALTH RELEVANCE: Public health relevance. Bacteria can evolve rapidly to evade killing by antibiotics. This project will determine the genetic changes that happen over short time periods leading to antibiotic resistance. These changes include exchange of resistance genes among isolates and activation of genes by mobile genetic elements. Knowledge of the mechanisms and frequency of genetic change will help in the design of strategies to detect antimicrobial resistance and select the best therapy for treatment of infections.